Oxygen Sensing—From Bedside to Bench
Authors: M.J. Percy, Flores A. Zhao Q., C. Harrison, T.R. Lappin, P.H. Maxwell, M.F. McMullin, F.S. Lee
Journal: Journal of the American Society of Nephrology (2006)

The resolution of the sequence of the erythropoietin (EPO) gene and the availability of recombinant EPO for therapy have caused a true revolution in the management of the anemia of uremic patients. In addition, great progress has been made in the clarification of the unique mechanisms controlling the synthesis of EPO and of the molecular mechanisms of its action. Understanding the mechanisms of the regulation of EPO synthesis has contributed to the understanding of the broader issue of cellular adaptation to hypoxia (1). Von Hippel-Lindau (VHL) disease, a rare genetic condition (2), and discovery of the mutated causal protein in this condition (3) have greatly helped clarify the mechanisms of regulation of EPO synthesis and issues beyond, not the least of which is renal carcinoma formation. The clarification of the molecular basis of the different forms of polycythemia (or erythrocytosis) (4–6) is another impressive confirmation of the concept of Sir Archibald Garrod: when analyzing alkaptonuria (7), he had stated with clairvoyance, much ahead of his time, that when one has understood the rare, genetically determined “inborn errors of metabolism,” one will also obtain insights into the molecular genesis of common polygenic diseases, as we would say today. To understand the following it is useful to briefly recapitulate the molecular control of EPO synthesis (8). As part of the defense reactions against hypoxia, the transcription complex hypoxia-inducible factor-1 (HIF-1) controls EPO gene expression, mainly in the kidney, as well as ubiquitous hypoxia defense reactions in all mammalian cell types (1). The HIF-1 complex contains a constitutively expressed β subunit and the HIF-α subunits HIF-1α or HIF-2α, the latter of which are regulated at the posttranslational level in an oxygen-dependent manner. How does oxygen regulate the concentration of the HIF complex? The HIF system is primarily regulated not by modulating the synthesis, but mainly by regulating the breakdown via the proteasome pathway in an oxygen-dependent manner. In a two-step process, the HIF molecule is first given the Judas kiss, so to speak, by molecular oxygen reacting with a prolyl residue, which then permits in a second step interaction with and capture by the VHL factor. VHL in the HIF/VHL complex is the substrate recognition unit of a proteasomal E3 ubiquitin ligase complex, which destines the marked molecule to proteasomic destruction. Consequently, the HIF-1α protein is stable under hypoxic conditions, but is degraded with exquisite rapidity under normoxic conditions so that its half-life is one of the shortest of any protein. The site-specific addition of oxygen to prolines of the α-subunits of HIF is catalyzed by proline hydroxylases, which are 2-oxoglutarate–and ascorbate-dependent and have bivalent Fe++ at the active center. Three proline hydroxylase isoforms have been identified, PHD1, PHD2, and PHD3, which are variably expressed in different tissues. Knockdown experiments with small interfering RNA showed that the three isoforms contribute in a nonredundant manner to the regulation of the HIF-1α and HIF-2α subunits, have different selectivities for the different hydroxylation sites within HIF-α subunits, and react differently to stimulatory signals (9). Experiments selectively silencing the PHD2 isoenzyme using short interfering RNA proved that this maneuver is sufficient to stabilize and activate HIF-1α under normoxic conditions in all human cells investigated, thus imitating the effect of hypoxia (10). In reticulocyte lysates, PHD2 is the most abundant isoform (9,11), which is also closely related to the ancestral proline hydroxylase in Caenorhabditis elegans (12). All these points are relevant for the selection of therapeutic targets discussed below. The above paper by Percy et al. (6) concerns a family with polycythemia (erythrocytosis). It was recognized long ago that—apart from polycythemia vera, a malignancy characterized by excessive proliferation of erythroid, myeloid, and megakaryocytic elements in the bone marrow—there are rare cases of familial polycythemia (or erythrocytosis), for which mutations in the EPO receptor have been made responsible, resulting in hyperresponsiveness to EPO and low EPO concentrations (4). A widely discussed case concerned the Finnish cross country skier Eero Mäntyranta, who won 4 gold medals, 1 silver medal, and 2 bronze medals in the 1960, 1964, and 1968 winter Olympics as well as several world championships. He had admitted to doping with amphetamine, but later it had also been shown that he had a familial form of autosomal dominant erythrocytosis characterized by increased sensitivity to EPO (13), thought to result from a gain of function mutation of the EPO receptor (4). He had hemoglobin concentrations >20 g/dl since childhood (13). This case, as well as other genetic conditions impacting athletic performance (14), raised the hotly debated ethical issue of whether it is fair to let genetically unprivileged athletes compete with such genetically privileged competitors (15,16), as well as concerns about gene doping (17). One interesting, endemic, autosomal recessive form of polycythemia, presumably resulting from a single founder in the distant past, had been observed in the Chuvash Autonomous Republic of the Russian Federation in the mid-Volga River region (5), and also, more recently, in southern Italy (18). Meanwhile, sporadic cases have been observed as well (19,20). Customarily one distinguishes primary polycythemias (i.e., inherited or acquired mutations of erythroid progenitor cells rendering them hypersensitive to EPO and causing downregulation of EPO concentrations in the circulation) from secondary polycythemias (i.e., caused by extrinsic factors such as elevated EPO concentrations with normal EPO responsiveness of progenitor cells). Interestingly, the Chuvash polycythemia is characterized by features of both primary and secondary polycythemia, i.e., hypersensitivity of erythroid precursors to EPO and elevated EPO concentrations. A recent paper identified homozygosity with respect to a missense mutation in the VHL protein due to an Arg200Trp substitution as the molecular basis of both the Chuvash polycythemia (21) and the sporadic form of the disease (19), although conceivably compound heterozygotes might have an analogous presentation (20). In the VHL syndrome, mutations of VHL are characterized by hemangioblastomas, pheochromocytomas, and renal cell carcinomas. Although the mutation in the Chuvash polycythemia concerns the VHL protein, such features are not seen, suggesting that either VHL has additional functions unrelated to the HIF system or alternatively that the Chuvash mutation causes a milder dysfunction of the VHL protein, which causes only decreased interaction between HIF-1α and VHL, reduced degradation of HIF-1α, and elevated HIF-1α concentrations under normoxic conditions. As a result, despite the absence of hypoxia, the transcription of HIF-1α–dependent genes is increased, particularly EPO, causing polycythemia, but also—without clinical sequelae affecting the phenotype—other HIF-1α–dependent genes such as glucose transporters, transferrin, transferrin receptor, and vascular endothelial growth factor. It had long been recognized, however, that forms of familial polycythemia that fail to have mutations of the VHL gene exist (4), raising the issue whether genetic abnormalities of other proteins in the above cascade can produce polycythemia as well. The above observation of Percy et al. (6) now adds a new twist to this issue by identifying a form of genetically determined autosomal dominant polycythemia that is caused by the mutation of another player in the hypoxia response cascade, upstream of the VHL step, i.e., a mutation the PHD2 isoform of prolyl hydroxylase. The authors investigated a family with polycythemia of father, daughter, and son. They all had somewhat variable mildly elevated hemoglobin values with inappropriate failure of EPO concentrations to be suppressed. There were no features of VHL phenotype. Sequencing revealed in all three family members a heterozygous C to G change at base 950 of the coding sequence of PHD2, resulting in a proline to arginine exchange at codon 317. It had been suspected that this proline is crucial for the coordination of the Fe++ ion at the active site. Of obvious interest are the functional consequences of this mutation. Without going into details, mutant PHD2 bound more weakly to HIF-1α than wild-type PHD2. Does less binding translate into less hydroxylation of HIF-1α and HIF-2α? In a proline hydroxylase assay in the presence of the necessary cofactors (2-oxoglutarate, ascorbic acid), the HIF hydroxylase activity of the mutant PHD2 was significantly diminished and it was also less able to suppress the HIF-1α or hypoxia-induced activation of a reporter gene. This rare and, clinically speaking, not extremely important form of familial polycythemia is of great interest also to nephrologists and is informative in several respects. First, as outlined above, PHD2 is the key PHD isoform that regulates HIF under physiologic conditions. Of interest with respect to erythropoiesis, it is also the isoform that is most abundant in reticulocyte lysates (9,11). PHD2 also appears to be the weakest link in the chain of the hypoxia response cascade, as apparently near-haploinsufficiency for PHD2 in the EPO-producing cells of these patients was sufficient to cause polycythemia, suggesting that in these cells there is not much PHD2 reserve capacity, making this an obvious target for intervention. This raises the issue whether pharmacologic inhibition of this weak spot would make sense. Of the three major factors necessary for proline hydroxylation, i.e., ascorbate, Fe++, and 2-oxoglutarate, 2-oxoglutarate appears to be an obvious candidate. Indeed, pharmacologic inhibition by what presumably is an analog of this cofactor (22) and can be administered orally has been tried successfully in a small number of patients with renal failure who responded with a significant increase of Hb concentration (23). Apart from pharmacokinetics and potential toxicity, the issue now is long-term safety, as stabilization and upregulation of HIF cause a wide spectrum of reactions (1), of which increased angiogenesis (24) and tumor growth (22) are theoretically of greatest concern. It is here that both the observations in Chuvash polycythemia (21) and particularly the polycythemia of PHD2 malfunction (6) is somewhat reassuring, as no major clinical problems, particularly no malignancies, were seen with the exception of esophageal carcinoma in the propositus of the paper by Percy et al. (6), which in a smoker might have another explanation. It should not give the nephrologist a headache whether low molecular weight inhibitors, presumably difficult to detect in urine samples that are not strictly timed, might further increase athletic performance in future Tours de France.